Many viruses evade host defense mechanisms by targeting specific host vulnerabilities, revealing critical points in host pathways regulating antiviral responses. Recent advances in our understanding of how influenza virus escapes host countermeasures have exposed novel mechanisms of virulence that can be exploited pharmacologically. The NS1 protein of influenza virus, a major virulence factor, inhibits host gene expression and signal transduction required to mount innate and adaptive immune responses. In infected cells, NS1 is localized in the nucleus and the cytoplasm. Based on the function of NS1 as inhibitor of gene expression, we performed a high throughput screen (HTS) of 200,000 synthetic chemical compounds and identified novel inhibitors of NS1. We selected eight classes of novel compounds that significantly restored gene expression in the presence of NS1, and that also inhibited both influenza virus replication and host cell death. We propose that antagonists of either nuclear and/or cytoplasmic activities of NS1 will be novel inhibitors of viral replication and pathogenesis. We will combine chemical biology, cell biology, and virology to investigate the mechanisms by which the compounds we have identified inhibit influenza virus replication. We will characterize two families of small molecules that can serve as leads for molecular therapy. In addition, the proposed studies will provide new insights into mechanisms by which influenza virus evades host defense pathways that can be further targeted pharmacologically. We will pursue the following aims: Aim 1. To investigate structure-activity relationship (SAR) of NS1 inhibitors. Aim 2. To determine the activity of NS1 inhibitors on Antiviral Responses in vivo. Aim 3. To identify targeted pathways and mechanisms of action of compounds which inhibit NS1 function. In sum, these studies will likely reveal novel leads for antiviral therapies as well as provide information on novel mechanisms of viral-host interactions and pathways.